Accumulating evidence has indicated that high-order genome structures influence transcription and mediate critical co-transcriptional crosstalk among distinct DNA elements. The functional significance of a variety of types of chromatin loops (e.g. promoter-terminator, enhancer-promoter, and interchromosomal) in gene regulation during development and in cancer cells has been demonstrated recently. Essential factors from the transcription initiation and mRNA 3'-end cleavage and polyadenylation (poly(A)) machineries have been shown to form complexes to confer promoter-terminator gene looping. The general transcription factor TFIIB mediates a critical interaction network involving Ssu72 (a pol II C-terminal domain phosphatase) and Rna15 (the poly(A) RNA-binding subunit of the cleavage factor IA complex). Gene looping is a widespread phenomenon in yeast and has also been identified for mammalian genes, plant genes, and human immunodeficiency virus provirus. In addition, gene looping has been connected to a number of fundamental cellular processes, which include transcription reinitiation, maintenance of transcriptional memory, and control of transcriptional directionality. In the proposed research, we will address the following key questions: (1) how do TFIIB and TFIIB- centered gene looping complexes regulate transcription (Aim 1), (2) what is the structural and biochemical basis for the pol II transcription-coupled gene loop establishment and maintenance (Aim 2), and (3) what are the mechanisms and physiological consequences of gene looping-dependent pol II reinitiation (Aim 3)? Our broad hypothesis is that unique macromolecular complexes formed by nuclear factors and non-coding RNAs mediate gene looping and create unique local chromatin environments to control pol II transcription. Specifically, we will test this hypothesis by (1) biochemically and structurally mapping binding interfaces of the TFIIB-centered gene looping complexes and connect them to the gene looping process directly in vivo, (2) revealing the pathways of TFIIB-dependent gene loop formation during the course of transcription and identifying additional nuclear factors (e.g. cohesin, condensin, nuclear pore complexes, and TFIIIC) required for gene looping, and (3) determining pol II and transcription factor dynamics on chromatin governed by gene loops with tools including the competition ChIP, the nuclear anchor-away system, and the chemically induced artificial gene looping system. Collectively, built upon our preliminary results these studies will provide the molecular mechanisms of gene looping and its mediated co-transcriptional crosstalk. In addition, we will link gene looping to other cellular pathways by identifying additional nuclear factors critical for this process. Finally, our studies may offer insights into the common principles of all types of transcription-dependent chromatin loops implicated in development and human disease and hence suggest novel targets for therapeutics.